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Mechanisms and Controls of DNA Replication in Bacteria

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 13

Mechanisms and Controls of DNA

Replication in Bacteria
César Quiñones-Valles, Laura Espíndola-Serna
and Agustino Martínez-Antonio
Departamento de Ingeniería Genética, Cinvestav Irapuato
México

1. Introduction
DNA is the polymeric molecule that contains all the genetic information in a cell. This
genetic information encodes the instructions to make a copy of itself, for the cellular
structure, for the operative cellular machinery and also contains the regulatory signals,
which determine when parts of this machinery should be on or off. The operative machinery
in turn, is responsible for the cells functions either metabolically or in interactions with the
environment. Part of this cellular machinery devoted to DNA metabolism is responsible for
DNA replication, DNA-repair and for the regulation of gene expression. In this chapter we
will focus our discussion on the mechanisms and controls that conduct DNA replication in
bacteria, including the components, functions and regulation of replication machinery. Most
of our discourse will consider this biological process in Escherichia coli but when possible we
will compare it to other bacterial models, mainly Bacillus subtilis and Caulobacter crescentus as
examples of organisms with asymmetrical cell division.
In order to maintain a bacterial population it is necessary that cells divide, but before the
physical division of a daughter cell from its mother, it is necessary among other check
points, that the DNA has been replicated accurately. This is done by the universal semi-
conservative replication process of DNA-strands, which generates two identical strand
copies from their parent templates. To better understand this process it has been divided
into three phases: initiation, elongation and termination of DNA replication. In each of these
steps, multiple stable and transient interactions are involved and we have summarized them
below.

2. Components and mechanisms of the general process of DNA replication

Bacteria are subject to sudden changes in their surroundings, so they have adapted diverse
strategies to allow them to persist through time. One of the adaptive changes consists in
modifying growth rates, which is accompanied by adjusting mechanisms that control the
timing of the cell-cycle. This adjustment ensures that the process of cell division is
coordinated with the doubling of cell-mass and with the proper replication and segregation
of the chromosome. The study of the cell-cycle in bacteria is usually divided into three
stages: the period between cell-division (cell birth) and the initiation of chromosome
replication, the period required to complete DNA replication (elongation of DNA) and, the
220 Fundamental Aspects of DNA Replication

final phase, which goes from the end of DNA replication until the completion of cell-
division (Wang & Levine, 2009).
Under the best growing-conditions, DNA replication starts immediately after cell division in
most cells (Wang et al., 2005). Since replication of the chromosome takes more time than that
the necessary for cell division under optimal culture conditions, such as E. coli growing in
rich media, at 37ºC with good aeration, it can happen that more than one event of DNA
replication can occur per cell cycle (Zakrzewska-Czerwinska et al., 2007). For the purposes
of this work we shall divide the DNA replication process in bacteria into three steps:
initiation, elongation and termination as follows.

2.1 Initiation of DNA replication

In bacteria, the process of DNA replication initiates in a specific DNA region called ““origin
of replication”” (ori) where multi-protein complexes are positioned and recruits additional
initiator proteins to form the Pre-Replicative complex (pre-RC) whose main function is
to facilitate the aperture of duplex DNA to permit the loading of the replicative DNA
helicase. The activity of this DNA helicase assists the entrance and assembly of a large
multi-subunit molecular machine, the replisome (Zakrzewska-Czerwinska et al., 2007; Ozaki
& Katayama, 2009).

2.1.1 oriC and its cis regulatory regions

The origin of replication in E. coli (oriC) is a small DNA sequence of about 245 bp (Figure 1),
which contains three AT-rich repeats named L, M, and R for left, middle and right positions
respectively, each 13 bp long (Hwang & Kornberg, 1992). The oriC region also contains
multiple boxes of 9 bp each where DnaA (replication initiation factor) proteins bind. These
DnaA boxes recruit DnaA in two forms; DnaA-ATP and DnaA-ADP, although they show
more affinity for the first form, which is the active replication initiation complex of DnaA.
There are three DnaA-boxes of high affinity named R1, R2 and R4 and seven of low affinity
(I1, I2, I3, T1, T2, R5M and R3), (Katayama et al., 2010; Ozaki & Katayama, 2009). The oriC
region also contains GATC DNA motifs dispersed throughout, the GATC motif is
recognized as a target for DNA-methylation by the Dam enzyme (DNA adenine
methyltransferase). Finally, the oriC region also has DNA-binding sites for the union of
several regulatory proteins such as Fis (Factor for inversion stimulation) and IHF
(integration host factor), which assist in bending the DNA at this region (Leonard &
Grimwade, 2009).
The comparison of the DNA sequence used as origin of replication in E. coli versus genomes
of other sequenced bacteria indicates that the nucleotide composition and size of these
regions is similar (Bramhill & Kornberg, 1988). A database of ori regions in bacterial
genomes, the DoriC database, which contains a compilation of known and predicted DNA
origins of replication in bacteria has been developed (Gao & Zhang, 2007).

2.1.2 DnaA is the key protein required to form the pre-RC

The critical step for the successful replication of DNA is the unfolding of the DNA strands at
the oriC region, action that is assisted by the orisome (proteins-oriC complex) (Leonard &
Grimwade, 2005). This complex mainly comprises of the activity of the initiator protein
DnaA. This protein belongs to the ubiquitous AAA+ superfamily of ATPases (ATPases
Mechanisms and Controls of DNA Replication in Bacteria 221

Fig. 1. Description of the origin of replication in the E. coli chromosome. The origin of
chromosomal replication (oriC) contains three AT-rich repeats (L, M and R), each 13
nucleotide residues long and multiple DnaA-binding sites. There are three higher-affinity
DnaA-boxes R1, R2 an R4 (dark blue) and seven lower-affinity sites Ǖ1, Ǖ2, I1, I2, I3, R5M and
R3 (light blue). All the DnaA-boxes preferentially bind DnaA-ATP rather than DnaA-ADP
complexes. oriC also contains one site where IHF binds (green), one for Fis (gray) and GATC
sites (orange) which are recognized by the Dam enzyme.
associated with a variety of cellular activities). The X-ray structure of crystals of this protein
from Aquifex aeolicus shows that the protein has four distinctive domains (Erzberger et al.,
2002). Domain I serves for the interaction with other proteins, among those identified are:
the replicative DnaB helicase and the DnaA-binding assistance protein DiaA (DnaA-initiator
association). Domain II is a flexible linker, which provides free rotation for the adjacent
domains III and I. Domain III has typical motifs that are characteristic of the AAA+ protein
superfamily of ATPases characterized by a conserved nucleotide phosphate-binding motif,
named Walker A (GxxxxGK[S/T]), where x is any amino acid residue). This domain serves
in protein binding to either ATP or ADP. When DnaA binds ATP it can form multimeric
structures each consisting of 5––7 protomers (DnaA-ATP) by interactions of one subunit with
the ATP of the anterior subunit through their ““arginine fingers”” as shown in Figure 2. It is
suggested that the DnaA-oriC complex forms a circular pentamer, which is stabilized by
interactions between each DnaA unit as mentioned before. The formation of these
complexes promotes the unwinding of DNA strands on the initiation of replication. Finally,
domain IV of DnaA has a helix-turn-helix motif that allows it to interact with the DnaA-box
of oriC (Figure 2), (Erzberger et al., 2002; Ozaki & Katayama, 2009).

2.1.3 Additional components of the orisome

There are additional components of the orisome that may increase or impede the further
unfolding of DNA at the origin of DNA replication. Some of these proteins in E. coli include
the histone-like DNA-binding proteins IHF and Fis. IHF is a protein that binds to DNA at a
poorly defined sequence. It stimulates the initiation of replication in vivo and in vitro. IHF
assists the binding of DnaA to the low-affinity DnaA-boxes during the formation of the pre-
replicative-complex. Contrarily, Fis seems to act as a repressor of initiation of DNA
replication by inhibiting the binding of DnaA and IHF to their targets sites on DNA. This is
achieved because Fis binds to oriC in a specific region of 13 nucleotides from position 87 to
222 Fundamental Aspects of DNA Replication

Fig. 2. DnaA as the main protein for the unfolding of DNA strands at oriC. A) The DnaA
protein family is part of the AAA+ ATPases. In E. coli DnaA contains four functional
domains as shown in the diagram. The ATP molecule is shown in red, and the arginine
finger in purple. B) Domain III binds preferentially ATP over ADP, in addition it has an
““arginine finger”” which permits the multimerization of these protomers over the DNA.
119 (Figure 1), (Cassler et al., 1995; Ryan et al., 2004). Additional proteins such as DiaA and
HU (Heat unstable protein) bind to domain I of DnaA, contributing to the stabilization of
the joining of their protomers to oriC (Ishida et al., 2004; Chodavarapu et al., 2008). Another
protein, ArgP (arginine protein, also called IciA) binds to the AT-rich regions in L, M and R
boxes blocking the opening of DNA by DnaA (Hwang et al., 1992), this protein binds in the
order of 10-20 monomers per oriC. Mutants in this gene however have no a clear defective
phenotype of DNA replication and possibly this protein is functioning as an additional
mechanism to maintain the robustness of this process. ArgP is also a transcriptional
regulator which counts dnaA among its target genes. The activity of ArgP is regulated
by arginine as its alosteric ligand and the protein is degraded by a specific protease. Another
protein that inhibits the binding of DnaA to its target sequences is CNU (oriC-binding
nucleoid-associated). CNU is a small protein composed of 71 amino acids (8.4 kDa)
that binds to a sequence of 26 bp (named cnb), which overlaps with the binding sites
for DnaA, thereby preventing its binding to oriC (Kim et al., 2005). When DnaA-ATP binds
to oriC it twists the DNA and promotes the separation of DNA-strands in the AT-rich region
to produce a single-stranded bubble or ““open complex”” (Figure 3). The next step is
the recruitment of the (DnaBC)6 complex to DnaA to obtain the pre-Replicative Complex
preRC.
Four or five DnaA-ATP molecules interact with the (DnaBC)6 complex via the N-terminal of
the replicative DnaB helicase and their common binding to oriC (Seitz et al., 2000). DnaB6 is a
monohexameric helicase with a ring shape. Its function is the unwind of double-stranded
DNA employing the hydrolysis of ATP, this activity is maintained as the elongation phase
proceeds. DnaB6 in its inactive form is found associated with the small protein DnaC (also of
the AAA+ superfamily) forming a closed complex DnaB6-(DnaC-ATP)6, (Biswas & Biswas-
Fiss, 2006).
The DnaB protein should be loaded onto each of the single-stranded DNA (ssDNA)
molecules. For this to happen, the pre-RC needs to release the DnaC from the complex
(DnaBC)6. It has been suggested that the DNA helicase translocates between parental
templates of DNA and interacts via its N-terminal domain with the DnaG primase. The
Mechanisms and Controls of DNA Replication in Bacteria 223

formation of the DnaB-DnaG complex is known as the ““primosome””. Since replication is

bidirectional in most bacterial chromosomes, one primosome is loaded on each single
stranded parental (Figure 3). DnaB is responsible for the unwinding of the double stranded
DNA (dsDNA) in the 5’’-3’’ direction and the primase synthesizes a small fragment of RNA
complementary to the parental DNA-strand, not shorter than 12 and up to 29
ribonucleotides (Figure 3), (Swart & Griep, 1995; Rowen & Kornberg, 1978). The interaction
of the primase with DnaB and the use of these primers trigger the release of DnaC. This
action defines discrete events in the transition from initiation to the elongation phase of
DNA replication (Makowska-Grzyska & Kaguni, 2010).

2.2 Elongation of DNA

Since the holoenzyme DNA polymerase III (Pol III, see below for components) cannot
initiate DNA polymerization de novo, the strands are extended from the RNAs synthesized
by the DnaG primase (Figure 4).
Pol III is positioned at the 3´ end of the first RNA primer complementary to the leading
strand of DNA and extends it continuously. In contrast on the lagging strand the new DNA-
strand is synthesized discontinuously producing Okazaki fragments of about 1 kb in length.
The RNA primers are removed and substituted by DNA by DNA polymerase I (Pol I). Pol I
uses 5´-3´exonuclease activity to remove these primers and fill out the gaps with its 3’’-5’’
DNA polymerase activity. Then DNA-ligase joins adjacent DNA fragments by catalyzing
the formation of phosphodiester bonds between the 5’’ phosphate of a hydrogen-bonded
nucleotide and an adjacent 3’’ OH of the nucleotide of the following Okazaki fragment.
The Pol III holoenzyme is composed of three subassemblies: the core polymerase, the ǃ-
sliding clamp and the clamp-loader complex. The core DNA polymerase is in turn,
composed of three subunits ǂ, lj and dž. The ǂ-subunit is that which really has the activity of
DNA polymerase whereas the small subunit dž has proofreading 3’’-5’’ exonuclease activity
and its function is to remove nucleotides that have been misincorporated by the core-
polymerase. The dž-subunit is stabilized by the lj-subunit, which as yet has not been assigned
additional functions (Schaeffer et al., 2005).
The clamp-loader or DnaX complex consists of six different subunits (Dž‘‘, Dž, DŽ, Ǖ, Ǚ, ǘ). DŽ and Ǖ
subunits are encoded by the same dnaX gene. The full sequence of dnaX encodes the protein
Ǖ. However when the mRNA is being translated the ribosome sometimes undergoes a frame
shift and a shorter product (only two-thirds) results. The frameshift occurs in a poly(A) tract
and yields a new stop codon immediately following the frameshift signal. This truncated
form of Ǖ corresponds to the DŽ protein. In this way, the first three domains of DŽ and Ǖ are
identical. These different protein versions bind to the Dž and Dž’’ subunits forming a complex
composed of Dž’’DŽDžǕ2 subunits. The ǘ-Ǚ dimer binds either DŽ or Ǖ subunits via the amino-
terminus of Ǚ constituting the clamp-loader (Gao & McHenry, 2001; Reyes-Lamothe et al.,
2010). Ǖ proteins have two defined interactions; on one side they attach to the ǂ-subunit of
the core and on the other, interact with the DnaB6 helicase on the lagging strand, so that this
complex forms a bridge between the replicase and helicase proteins (Lee et al., 1996).
The single strands of DNA (ssDNA) are stabilized by a protein called single-stranded
DNA-binding protein (SSB). SSB binds to single DNA-strands as a tetramer through its N-
terminal domain, which makes contact with the DNA. The clamp-loader recognizes
ssDNA coated by SSB4, interacting with the ǘ subunit of SSB4. ǘ forms a heterodimeric
complex with Ǚ, which in turn, interacts with the DŽ and Ǖ subunits. In this way ǘ senses
the presence (or absence) of ssDNA, facilitating the recognition of the terminal parts of
RNA primers by Ǖ (Schaefffer, 2005).
224 Fundamental Aspects of DNA Replication

Fig. 3. Formation of the pre-RC. A) Binding of DnaA-ATP to oriC to form multimeric structures
in conjunction with DiaA and (DnaBC)6 via domain I, these interactions are important in order
to form the pre-RC. B) The binding of DnaA-ATP to this region of DNA is favored when the
protein IHF also binds to oriC, about 20 molecules of DnaA-ATP bind to OriC simultaneously.
This DnaA-ATP complex is stabilized by DiaA and finally leads to the unfolding of the DNA
at the AT-rich region. At this stage the (DnaBC)6 complex is attached to domain I of the DnaA-
ATP, forming the pre-RC. Subsequently, DnaB releases DnaC and loads to each single
stranded DNA in direction 5’’-3’’ with the assistance of the DnaG primase.
The sliding-clamp (ǃ2) is a dimer of DnaN proteins, which binds to the hybrid DNA-RNA
and serves to direct Pol III to this position for the synthesis of Okasaki fragments. During
the elongation phase Pol III can hop from one clamp to another without leaving the
replication fork. So Pol III overcomes possible delays due to blockage of DNA by the activity
of transcription factors or DNA damage (Georgescu et al., 2010).

2.3 Termination of DNA replication

The end of DNA replication takes place when the replisome helicase DnaB6 on the leading
strands collides with a protein called Tus. Tus recognizes and is bound to sites for
termination of DNA replication (ter). These sites are physically arranged in positions
opposite to the oriC (Figure 5). In the collision of Tus with the helicase a trap is formed that
prevents the further advancement of the replicative machinery in the leading strand and
remains arrested until the replicative machinery on the lagging strand reaches this position
(Neylon et al., 2005).
Mechanisms and Controls of DNA Replication in Bacteria 225

Fig. 4. Elongation of DNA by the replisome machinery.

The elongation of DNA in the E. coli chromosome is carried out in both directions of the fork
by a multisubunit machinery called the replisome. Each replisome is located in both directions
of the fork. The helicase DnaB6 is loaded on the (3’’-5’’) lagging strand to unfold the DNA
duplex in the 5’’-3‘‘ direction, at this time the primase synthesizes RNA primers complementary
to each ssDNA. These primers are extended by Pol III, forming the Okasaki fragments on the
lagging strand. When Pol III extends a new Okasaki fragment and reaches a previously
synthesized one, it gives a hop, joining to another slider clamp (ǃ-subunit), which recognizes
DNA-RNA hybrids. DNA polymerases working on both parent strands are coordinately
driven by the clamp-loader, which also binds to the helicase. SSB stabilizes the ssDNA. For the
recognition of ssDNA by Pol III, the clamp-loader makes contact with SSB4-DNA via its ǘ-
subunit.
The resolution of chromosomes is produced by the activity of several proteins which act
together to separate the two daughter chromosomes. In this process the FtsK protein is very
important as it acts by coordinating cell division with chromosome segregation through the
activities of its N-transmembranal domain (FtsKN) and its C-cytosolic domain (FtsKC),
respectively. FtsKN is the target for the division protein that forms the septum FtsZ, which
stabilizes the interactions of FtsK with the other components of the divisome FtsQ, FtsI and
FtsL (Aussel et al., 2002; Dubarry et al., 2010). FtsK also contains a linker, FtsKL, localized
between the FtsKN and FtsKC domains (Bigot et al., 2004). Recently two distinct regions
within FtsKL; have been identified (FtsK179––331 and FtsK332––641), which together with
FtsKN, are required for normal septation in E. coli (Dubarry et al., 2010). FtsKC can lead to the
dimerization of circular chromosomes, thereby compromising their segregation (Figure 5).
FtsKC activates events of recombination at the dif site (localized beside the replication
termination region), which are mediated by two proteins with activities of tyrosine
recombinases, XerC and XerD to resolve chromosome dimers to monomers and at the same
time promote DNA translocation (Bigot et al., 2004; Kennedy et al., 2008). FtsKC is part of the
226 Fundamental Aspects of DNA Replication

AAA+ superfamily and therefore can form a ring-shaped multimer that wraps the DNA and
moves along it at the expense of ATP. When a chromosome dimer is present, a site-specific
recombination event by XerCD introduces an additional cross over at dif, resolving thus the
dimer into two monomers, all this is under the control of FtsK (Aussel et al., 2002).

Fig. 5. Termination of DNA replication. A) The site of termination of replication in E. coli is

opposite to oriC, where there are specific. ter sequences which are recognized by the Tus
protein (purple boxes). B) Tus protein-terminator sequence (Tus-ter) is a barrier that pauses
the leading fork until the lagging fork arrives from the opposite direction and induces
termination, which occurs when the helicase touches Tus. The helicase dissociates from
DNA and Pol III synthesizes the complementary strand on both sides of the forks. C) Near
to the Tus-ter sites is found a sequence named dif, where site-specific recombination
mediated by the XerC and XerD recombinases assisted by the translocase FtsK takes place.
Figure taken and modified from Aussel et al. (2010).
A summary of the key enzymes involved in DNA replication known to date in Escherichia
coli, are shown in table I.

3. Regulation of DNA replication

The regulation of DNA replication is a vital cellular process. In a general view, DNA
replication is controlled by a series of mechanisms that are centered on the control of cellular
DnaA levels, its availability as a free protein and modulation of its activity by binding the
small-molecule ligand ATP (Leonard & Grimwade, 2009); the other point of control is by
modulating the accessibility of replisome components to the oriC region on the DNA. We
discuss some aspects of these regulatory mechanisms below.
Mechanisms and Controls of DNA Replication in Bacteria 227

Gene
Protein Gene MWa
Function length Essentialityb
name name (kDa)
(bp)
Initiator of DNA synthesis by binding to the origin of
replication and also acts as a transcriptional
regulator. It binds to DnaA boxes, and binds ATP.
DnaA dnaA 1404 52.551 E
Around 20 to 30 DnaA monomers bind to the oriC
region. It is calculated that around of 1000 molecules
per cell are bound reaching up to 70% DnaA-ATP.
A hexameric DNA helicase, it progressively unwinds
DnaB dnaB DNA strands ahead of replication forks. About 100 1416 52.39 E
DnaB molecules are calculated to be present per cell.
DnaC is an accessory protein that assists the loading
of DnaB onto DNA duplex to initiate replication and
DnaC dnaC onto ssDNA to assist primer formation by the 738 27.935 E
primase. Six DnaC monomers bind to the hexameric
DnaB
DNA primase, it catalyzes the synthesis of RNA
primers on ssDNA. These primers are necessary for
DNA synthesis by DNA polymerase III. A DnaB––
DnaG dnaG DnaG complex was observed by mixing DnaB with a 1746 65.565 E
six molar excess of DnaG (hexamers of DnaB and
monomers of DnaG). Log-phase cells contain 50 to
100 molecules of primase.
DNA polymerase III holoenzyme is the primary enzyme for DNA synthesis in E. coli.
DNA It carries out 5’’ to 3' DNA polymerization using ssDNA as a template; it also carries
polymerase III out 3'-5' exonuclease edition of mispaired nucleotides. There are estimated to be 10
holoenzyme holoenzymes of DNA polymerase III per cell.
(Pol III) Pol III holoenzyme is made up of the following components
[(DnaE)(DnaQ)(HolE)]3[(DnaX)3(HolB)(HolA)][(DnaN)2]2[(DnaX)2][(HolC)(HolD)]4.
DNA
The DNA polymerase III core enzyme can carry out the basic polymerase and
polymerase III
exonuclease activities of polymerase III.
(core)

ǂ dnaE ǂ subunit catalyzes DNA polymerization from 5' - 3'. 3483 129.9 E

dž dnaQ dž subunit catalyzes the 3' - 5' proofreading activity 732 27.099 E

lj holE lj subunit allows stabilization of ǂ and dž subunits 231 8.846 NE

The ǃ subunit dimerizes to form the sliding clamp

ǃ dnaN 1101 40.587 E
which positions the core polymerase onto the DNA.

It catalyzes ATP-driven assembly of the sliding clamp onto primer-template DNA.

Clamp loader
Clamp loader = DžDž’’Ǖ2DŽǙǘ
Dž subunit acts as a wrench to open the sliding clamp
probably using ATP. Some Dž units exist
Dž holA independently of the preinitiation complex, possibly 1032 38.704 E
playing a role in stripping ǃ clamps from DNA in the
absence of replication initiation.
228 Fundamental Aspects of DNA Replication

Dž' holB Dž' subunit is part of the clamp loader complex. 1005 36.937 E

Ǖ subunit binds to the alpha subunit dimerizing the

Ǖ dnaX core alpha-epsilon-theta polymerase subunits. This is 1932 71.138 E
required for synthesis on the lagging strand.

DŽ dnaX DŽ subunit is part of the clamp loader complex. 1932 47.545 E

ǘ subunit allows the binding of the clamp loader to

ǘ holC SSB. Ǚ-ǘ also acts in multiple ways improving the 444 16.633 E
binding of DNA polymerase to DNA templates.
Ǚ subunit allows the interactions between DŽ and Ƹ
Ǚ holD 414 15.174 NE
subunits
Fis for "factor for inversion stimulation" allows the
organization and maintenance of the nucleoid
structure through direct DNA bending and by
modulating the production of gyrase and
Fis fis topoisomerase I as well as regulating the expression 297 11.24 E
of other proteins that modulate the nucleoid
structure, such as HNS, and HU. It reaches a cell
concentration of 40,000-60,000 molecules/cell at the
beginning of the exponential phase
The DNA adenine methyltransferase is responsible
for methylation of GATC sequences in E. coli. A wild-
Dam dam type, rapidly growing E. coli cell (doubling time = 30 837 32.1 NE
min) was found to contain about 130 molecules of
Dam methyltransferase.
DiaA interacts with DnaA, it is required for the
timely initiation of chromosomal replication and
DiaA diaA 591 21.106 NE
stimulates the replication of minichromosomes in
vitro.
The ArgP transcriptional activator or inhibitor of
chromosome initiation (IciA) regulates DNA
replication by binding to three 13-mers located in the
origin of replication (OriC), blocking the DNA
ArgP/Ic
argP opening by DnaA. It is also a transcriptional 894 33.472 NE
iA
repressor of dnaA. There are about 800
molecules/cell of IciA in the exponential phase and
the level decreases to about 500 molecules per cell in
the early stationary phase.
"Integration host factor", is a global regulatory protein that helps to maintain the DNA
architecture. It binds and bends DNA. IHF plays a role in DNA supercoiling and
DNA duplex destabilization and affects processes such as DNA replication,
IHF
recombination, and the expression of many genes. Consisting of two subunits ǂ and
ǃ. IHF reaches 6,000-15,000 complexes in the exponential phase and up to 30,000-
55,000 in the stationary phase.

IHF-ǂ ihfA ǂ subunit of IHF 300 11.354 NE

IHF-ǃ ihfB ǃ subunit of IHF 285 10.651 NE

Mechanisms and Controls of DNA Replication in Bacteria 229

HU for heat unstable protein, is a global regulatory protein and shares properties
with histones for nucleoid organization and regulation. It is a heterodimer formed by
HU
an ǂ- and a ǃ-subunit. HU reaches 30,000-55,000 dimers in the exponential phase and
10,000-17,000 in the stationary phase.

HU-ǂ hupA ǂ-subunit of HU 273 9.535 NE

HU-ǃ hup B ǃ-subunit of HU 273 9.226 NE

In addition to polymerase activity, this DNA

polymerase exhibits 3'ń5' and 5'ń3' exonuclease
DNA activities. It is able to utilize nicked circular duplex
polA 2787 103.12 NE
Pol I DNA as a template and can unwind the parental
DNA strand from its template. Its cellular abundance
is of around 400 molecules per cell.
LigA is one of two known NAD(+)-dependent DNA
LigA ligA ligases, it catalyzes the formation of phosphodiester 2016 73.606 E
bonds on duplex DNA.
Single-stranded DNA-binding protein acts as a
SSB ssb tetramer when binding to DNA. Each E. coli cell has 537 18.975 E
about 800 monomers of SSB.
Tus, also known as ter-binding protein (TBP), binds
Tus tus to ter sites, blocking the progress of DNA replication 930 35.783 E
in a polar like form.
FtsK is an essential cell division protein linking cell
FtsK ftsK 3990 146.66 E
division with chromosome segregation
Regulator of DnaA that prevents premature initiation
Hda hda of DNA replication. Around 100 molecules/cell are 702 28.37 E
found.
A RNA Polymerase-binding ATPase and RNAP
RapA hepA 2907 109.77 NE
recycling factor.
Sequesters newly replicated hemimethylated oriC to
SeqA seqA prevent re-initiation; it also binds hemimethylated 546 20.315 E
GATC sequences.
Xer site-specific Two lambda integrases of the family of recombinases involved in converting
recombination chromosome dimers of into monomers so that segregation of the chromosomes can
system occur during cell division

XerC xerC XerC is part of the Xer site-specific recombination 897 33.868 E
system

XerD xerD XerD is part of the Xer site-specific recombination 897 34.246 NE
system

a MW: Molecular weight of the polypeptide product.

b Essential gene (E)/ non essential gene (NE).

Table 1. Description of major proteins for replication in E. coli

230 Fundamental Aspects of DNA Replication

3.1 Regulatory mechanisms of DNA replication in E. coli

One of the main mechanisms associated with DNA replication is the so-called RIDA system
(Regulatory Inactivation of DnaA). The elements of this system are the sliding-clamp of
DNA polymerase III and Hda (Homologous to DnaA). This mechanism takes place when
DnaA is activated by its binding to ATP. The accumulation of DnaA in this active form leads
to the initiation of chromosomal replication since it facilitates its binding to the oriC on the
DNA. DnaA reverts to its inactive form DnaA-ADP by hydrolysis of ATP (Katayama et al.,
1998). Hda-ADP is the monomeric active form for promoting the hydrolysis of DNA-ATP, a
process which is mediated by the slider-loader clamp (Su’’etsugu et al., 2008). This
inactivating regulation of DnaA is key for preventing the over-initiation of replicative events
during the cell cycle (Katayama & Sekimizu, 1999). The free-living bacteria C. crescentus also
presents this regulatory mechanism, as it has HdaA, a protein similar to the E. coli Hda. In
C. crescentus HdaA also inactivates DnaA in a replication-coordinated manner, if DNA
replication is successfully initiated then HdaA and the ǃ-sliding clamp promote the
hydrolysis of DnaA-ATP to DnaA-ADP and force DnaA to leaves the oriC (Collier &
Shapiro, 2009). A conserved bacterial protein, YabA, has been found in B. subtilis and other
Gram-positive bacteria where it acts as a repressor for initiation of DNA replication. This is
achieved by forming a complex with DnaA and the ǃ-sliding clamp independently of the
DNA, a common activity shared between Hda and YabA (Mott & Berger, 2007). Thus the
RIDA system is present in B. subtilis and is also the primary mechanism for regulation of
DNA replication in this bacterium (Noirot-Gros et al., 2006). The formation of the oriC and
DnaA complex is assisted by the protein DiaA, which forms homo-tetramers and binds
various DnaA molecules, especially in the active form of DnaA-ATP but it can also stimulate
the formation of the DnaA-ADP-oriC complex, this is an inactive complex for initiation of
replication (Ishida et al., 2004).
Another mechanism that regulates the initiation of DNA replication is by controlling the
availability of free DnaA to bind to DnaA boxes on the oriC (Figure 1). Here the role of the
1kb datA locus, which is localized near (downstream) from the oriC is important. The datA
locus shows high affinity for DnaA, even more than the DnaA boxes on the oriC. Thus the
datA region is able to bind over 300 DnaA molecules whereas oriC binds to 45 DnaA
monomers (Kitagawa et al., 1998). The operability of this mechanism is facilitated by the fact
that the oriC had only few DnaA boxes compared to the datA locus and by the close
proximity of data in respect to oriC on the DNA molecule (Figure 6).
One related control system depends on the property of DnaA to act as a transcription factor
and to the presence of DnaA boxes in the promoter regions of several genes. In most cases
DnaA represses the expression of the associated gene but in some cases it can activates
certain genes (Messer & Weigel, 1997). DnaA regulates around 10 genes in E. coli as
documented in RegulonDB (Gama-Castro et al., 2010). The transcription of DnaA is one of
the most important regulatory mechanisms that directly affect the replication of DNA and
one of the proteins that negatively regulate the expression of dnaA is DnaA itself (Figure 6).
At high levels DnaA binds to the DnaA boxes in the promoter region and impedes
transcription. This auto-repressive process directly affects the amount of DnaA-ATP
available and controls the efficiency of initiation of DNA replication (Mott & Berger, 2007).
In C. crescentus, it was found that DnaA also auto-represses the transcription of its own gene
but additionally DnaA is highly unstable in this organism and gradually degrades after
initiating a replication event (Gorbatyuk & Marczynski, 2005).
Mechanisms and Controls of DNA Replication in Bacteria 231

Fig. 6. Mechanisms that regulate DNA replication in E. coli. A) The newly replicated DNA
duplex is in a hemimethylated state. B) SeqA binds to the hemimethylated GATC sites
immediately after they are replicated. C) RpoD activates the transcription of dam and Dam
methylates GATC sites of the newly synthesized strand. D) HU represses the transcription
of SeqA. E) DnaA binds to the DnaA boxes on the oriC region. F) when there are many
DnaA molecules they repress the transcription of the dnaA gene. G) datA locus binds many
DnaA molecules.

3.2 Regulation of DNA replication by DNA methylation

A requirement for initiation of DNA replication is that both DNA strands are methylated,
principally the adenine nucleotide in the GATC motifs, this process is mediated by Dam
(DNA adenine methyltranferase), (Wion & Casadésus, 2006). Dam binds to the DNA
nonspecifically, and methylates the GATC motifs (Figure 6). On DNA strands recently
synthesized these motifs are rapidly methylated and exist in the hemimethylated state only
during a fraction of the time needed for the replication of the entire DNA (Casadésus &
Low, 2006).
The methylation process occurs asynchronously on the newly synthesized strands; i. e.
methylation on the lagging arm occurs only after the ligation of the Okazaki fragments. It is
postulated that Dam is always present in a complex bound near the replication origin, thus
the methylation of nascent DNA strands occurs as soon as polymerization begins. In
summary, the presence of hemimethylated GATC sites provides a cue to indicate that DNA
replication has just occurred (Stancheva et al., 1999).
Another way to repress the transcription of dnaA is that which occurs immediately after the
initiation of DNA replication. Here, SeqA binds to the hemimethylated GATC sequences in
the regulatory regions of the dnaA gene (Lu et al., 1994; Brendler et al., 2000). Similarly, SeqA
also represses the replication of DNA by binding to the hemimethylated GATC sequence at
the oriC, this is possible because SeqA DNA-binding sites overlap with those of low affinity
for DnaA (DnaA boxes) on the oriC. This overlap impedes the complete access of DnaA-ATP
to the oriC (Han et al., 2004).
This prevention of replication, dependant of DNA methylation, has been considered as an
epigenetic regulatory mechanism because it depends on the chemical modification of the
nucleotide residues of the DNA and not in its sequence.

3.3 Regulation of DNA replication in Bacillus subtilis

B. subtilis shares some orthologous genes to the regulators that are involved in DNA
replication in E. coli, but particular regulatory mechanisms must occur in this organism, as it
232 Fundamental Aspects of DNA Replication

lacks some important components of the regulatory machinery found in E. coli such as the
seqA and dam genes. In their place other players are present in B. subtilis such as Spo0A
(Figure 7) and SirA (sporulation inhibitor of replication) (Katayama et al., 2010). Spo0A is
the master regulator for sporulation and, at the same time, is an inhibitor of DNA
replication. Spo0A is activated by a multicomponent phosphorelay process, this is initiated
by a histidine kinase (KinA), that autophosphorylates, and transfers the phosphate to Spo0A
through two intermediate phosphotransferases (Spo0F and Spo0B), (Burbulys et al., 1991).
Spo0A-P (the active form) binds to specific sites on the oriC region and blocks the
unwinding of the DNA duplex. Spo0A-P activates SirA, and SirA binds to DnaA in Domain
I inhibiting the ability of DnaA to bind to the oriC (Wagner et al, 2009). Sda maintains the
cellular levels of Spo0A-P low when a new round of replication has initiated (Veening et al.,
2009), by inhibiting the accumulation of the autophosphorylated form of KinA
(Cunningham & Burkholder, 2008).
Other regulators also implicated in DNA replication in B. subtilis are Soj and Spo0J (Figure
7), both components are required for proper chromosome segregation and for the repression
of DNA replication. Soj exerts its activity in repressing replication by interacting with DnaA
at the oriC, thereby preventing DnaA from initiating DNA replication (Murray & Errington,
2008). Otherwise Spo0J produces the complex Soj-Spo0J at the parS locus (Autret et al., 2001),
promoting the release of Soj from the DNA strands, and allowing DNA replication to be
initiated (Lee et al., 2003).

Fig. 7. Mechanisms that regulate DNA replication in B. subtilis. A) Soj represses DnaA
activity. B) Spo0J stimulates Soj binding to the parS locus. C) The complex of Soj at the parS
locus promotes the separation of Soj from the DNA. D) DnaA binds the DnaA boxes in the
oriC initiating DNA replication. E) DnaA represses dnaA itself and activates the transcription
of sda. F) Sda inhibits the accumulation of KinA-P. G) KinA activates Spo0A by transferring
a phosphate group to Spo0A. H) Spo0A binds to specific sites in the oriC and represses
replication, it also represses dnaA and spo0J and activates sirA. I) SirA, in turn, binds to
DnaA and represses its binding to the oriC.

3.4 Regulation of DNA replication in Caulobacter crescentus

An interesting mechanism for control of DNA replication takes place in the cell cycle of C.
crescentus, this aquatic, free-living bacteria, divides asymmetrically and this process is
Mechanisms and Controls of DNA Replication in Bacteria 233

regulated by a complex circuit of master regulatory proteins (Figure 8) coupled to a two-

component system.
One of these regulators is the master regulator of cell cycle CtrA (Cell cycle transcriptional
regulator), which is transcriptionally regulated by methylation of the GANTC motif on the
first of the two of ctrA promoters (P1). Transcription initiation at P1 is repressed when the
GANTC motifs are fully methylated while in the hemimethylated state transcription takes
place. This mechanism ensures that ctrA is transcribed only while replication is in progress,
producing enough protein to block and prevent the reinitiating of another round of DNA
replication during this time (Reisenauer & Shapiro, 2002). In the hemimethylated form the
production and accumulation of CtrA occurs, this protein binds to the regulatory region of
ccrM and activates the transcription of a DNA-methylase encoded by this gene. Once
synthesized, this enzyme proceeds to complete the methylation of both DNA strands. CtrA
ceases its repressing activity when it is degraded by a Lon-type protease. The transcription
of ccrM mediated by CtrA is inhibited when the two GANTC regulatory motifs are
methylated. This complex machinery determines that when DNA is fully methylated, the
transcription of ctrA and ccrM genes turns off (Stephens et al., 1995). This regulatory
mechanism ensures that the synthesis of CcrM remains off and takes place only when the
replication fork reaches the position of the ccrM gene preventing its premature transcription
(Reisenauer et al., 1999).
The phosphorylated state of CtrA (CtrA-P) is the active form of this regulatory protein and
this process is mediated by a cascade of phosphorylations which start with the activation of
Divk, mediated by CtrA. DivK transfers the phosphate group to the CckA intermediate (Cell
cycle histidine kinase) and CckA and ChpT finally transfer the phosphate group to CtrA. In
the swarmer cell type of C. crescentus, CtrA-P binds to five DNA motifs on the oriC region,
repressing the process of DNA replication (Marczynski & Shapiro, 2002). For the replication
process to take place CtrA-P must be degraded by the ClpXP protease, which releases the
origin of replication. ClpXP and CtrA are localized to each of the poles in stalked cells. This
polar targeting of ClpXP is mediated by CpdR (a two component receiver protein), which is
a dephosphorylating protein positioned at the pole where it recruits ClpXP (Jenal, 2009).
Sometime after this happens, the proteolysis of CtrA ends and a positive transcriptional
feedback loop generates the accumulation of CtrA, blocking again the access of DnaA to the
oriC (Hung & Shapiro, 2002).
Another regulatory system for DNA replication in C. crescentus, is the regulatory circuit of
DnaA, CtrA, GcrA and SciP. This genetic circuit regulates the transcription of multiple genes
(DnaA alone controls the expression of approximately 40 genes in this bacterium) and many
of these genes encode components of the replisome, in particular activating gcrA. On the
other hand, CtrA regulates about 95 genes principally those involved in flagella biogenesis,
cellular division and other regulators, and inhibits gcrA. GcrA in turn controls over 50 genes
including the activation of ctrA and the repression of dnaA (Laub et al., 2007). Finally, Scip
represses ctrA, and it is regulated in a feed forward loop manner; activated by CtrA and
repressed by DnaA (Tan et al., 2010).
Some of the regulatory mechanisms concerning DNA replication are conserved in bacteria
(as shown throughout this chapter) but specific mechanisms are also characteristic of each
organism, table 2 shows the comparison of the regulators present in the three bacterial
models described above.
234 Fundamental Aspects of DNA Replication

Fig. 8. Regulatory circuits that control the process of DNA-replication in C. crescentus. A)

CtrA activates and represses the transcription of its own gene, additionally it activates gcrA,
ccrM and sciP. B) CckA and ChpT transfer the phosphate group to CtrA. C) CtrA-P binds to
oriC and inhibits the initiation of DNA replication. D) SciP represses the transcription of
ctrA. E) DnaA auto-represses its own transcription in addition to the clpXP and sciP genes, it
also actives gcrA. F) ClpXP degrades both CtrA and CtrA-P forms. G) DnaA binds to oriC to
promote the initiation of DNA replication. H) CcrM methylates the GANTC sites on the
regulatory regions of dnaA, ctrA and on its own gene, and also on the oriC region. I) GrcA
activates the transcription of ctrA. Figure taken and modified from Tan et al. (2010).

4. The stringent response arrests DNA replication in bacteria

When bacteria are under metabolic stress, mainly in starvation conditions, they activate a
regulatory mechanism called the stringent response. This response usually corresponds to
Mechanisms and Controls of DNA Replication in Bacteria 235

Regulatory genes present in the organisms

Regulatory systems of
DNA replication
E coli B. subtilis C. crescentus

RIDA Hda YbaA * HdaA*

DnaA autoregulation,
dnaA gene regulation DnaA, Soj, SirA DnaA, CcrM
promoter methylation

DnaA-ATP/ADP, DnaA-
DnaA regulation DnaA-ATP/ADP
datA sequestration ATP/ADP

oriC blocking SeqA Spo0A CtrA

DNA methylation Dam - Ccrm

Phosphorylation KinA, Spo0F,

- DivK, ChpT, CckA
cascade Spo0B

Proteolysis - - ClpXP

* Orthologous to the E. coli components. - unidentified.

Table 2. Comparison of the controls that regulate DNA replication in E. coli, B. subtilis and
C. crescentus.
the deprivation of amino acids, carbon, and limitations of nitrogen and phosphate. Under
these conditions the cells suffer a reduction in size and restrict the content of their genetic
information to only one nucleoid per cell (Schreiber et al., 1995).
The signal which triggers the stringent response is mediated by the accumulation of small-
molecule nucleotides. These are guanosine tetra- and penta-phosphates; ppGpp and
pppGpp (Ferullo & Lovett, 2008). These alarmones are synthesized as a response to the
nutritional limitations by the proteins ReIA (synthetase I) and SpoT (synthetase II),
(Bernardo et al., 2006). During the stringent conditions the elongation phase of DNA
replication is inhibited because ppGpp and pppGppp specifically block the activity of the
primase enzyme (DnaG). This is caused by the binding of a phosphate group of ppGpp to
the primase resulting in an allosteric inhibition of the replication complex, the primase
cannot therefore bind to the helicase. High cellular levels (up to millimolar concentrations)
of these small nucleotides completely arrest DNA replication whereas lower levels only
diminish the rate of replication (Wang et al., 2007).
Another path of regulation of DNA replication under a stringent condition is produced by
the fact that the promoter of dnaA is also subject to the stringent response and the
transcription of dnaA is also repressed under these conditions (Chiaramello & Zyskind, 1990;
Levine et al., 1995).
236 Fundamental Aspects of DNA Replication

5. DNA replication and asymmetrical bacterial cell-division

In B. subtilis the arrest of DNA replication takes place around the oriC, from the gnt gene
on the left arm over an equal distance to the gerD gene of right arm, covering at least 190
kpb on both sides of the oriC (Levine et al., 1991). During this process the stages of
chromosomal segregation in cell division differ between prespores and vegetative cells.
First, the newly replicated chromosomes are attached at each of the cell poles (one pole
will become the spore and the other pole the mother cell). Upon the asymmetric septation,
under stress, about 30% of one of the replicated chromosomes is trapped in the prespore
(Wu & Errington, 1994). The protein SpoIIIE forms a pore in the invaginating septum
around the trapped DNA and permits the transfer of the remaining chromosome through
the septum into the prespore (Lewis, 2001). All this produces an imbalance among
regulators in the forespore and vegetative cell that results in an asymmetrical cell division
in B. subtilis.
Another example of asymmetrical cell-division happens in C. crescentus, this bacterium
differentiates into two different progeny: a flagellated swarmer cell and a stalked cell. The
swarmer cells are incapable of replicating their DNA (prevented by the mechanisms
previously mentioned in this chapter), until they differentiate into a stalked cell, this cell-
type immediately enters into a new period of chromosome replication and cell division, and
generates again the two cell types (Ryan & Shapiro, 2003). When C. crescentus is starved of
carbon sources, its DnaA protein is degraded in a manner that depends on the stringent
response mediated by the protein Spo, a ppGpp synthetase (Lesley & Shapiro, 2008).
Additionally starvation increases the degradation of DnaA leading to the stabilization of
CtrA resulting in the inhibition of DNA replication (Gorbatyuk & Marczynski, 2005).

6. DNA replication in bacteria with two chromosomes

Until now, in this chapter we have discussed replication focusing on bacteria with one
chromosome, but some bacteria have more than one chromosome, one example of this is
Vibrio cholerae, a human pathogen, which possesses two chromosomes, chrI and chrII
(Heidelberg et al., 2000). The components and regulation of DNA replication for chrI in V.
cholera are similar to the oriC of E. coli whereas the oriC of chrII shares some characteristics
with plasmid replicons. Both cases (chrI and chrII) also require a specific repeated sequence
for the replicative machinery (Zakrzewska-Czerwięska, et al., 2007). One of the specific
requirements is that chrI initiates replication assisted only by DnaA whereas chrII requires
the activity of the RctB protein that binds specifically to its oriC (Duigou et al., 2006), and an
untranslated trans-acting RNA (rctA) (Egan et al., 2005). However the two chromosomes
replicate synchronously although each has requirements for specific components which
reduces the competition between both origins of replication for the replicative machineries
(Duigou et al., 2006).
The proper regulation of DNA replication in bacteria with multiple chromosomes must
involve interesting strategies to control the replication of both chromosomes. Unfortunately
our knowledge about the regulation of DNA replication in these cases is poorly understood.
It has been suggested that organisms with two chromosomes have an advantage for
regulation of replication in some environmental conditions such as in free-living aquatic
conditions or in association with a host, since faster replication of all DNA content is
facilitated (Egan & Waldor, 2003).
Mechanisms and Controls of DNA Replication in Bacteria 237

7. Bacteria with multiple nucleoids

Another interesting phenomenon associated with DNA replication is endoreduplication
(duplication of DNA in the absence of cell-division) as happens in the differentiation of
Rhizobium etli, when these bacteria form a nodule and enter on it, in an endosymbiotic
association with roots of leguminous plants. Irreversible cell differentiation occurs in these
bacteria, which generates a nitrogen fixing bacteroid that is metabolically and
morphologically different from the original pre-nodule cell. The differences between these
types of cells result from cellular elongation and endoreduplication, without cell division.
These bacteroid cells result from normal cells suffering repeated rounds of DNA replication
and since the cell division is blocked they have multiple nucleoids (Mergaert et al., 2006).
Interestingly this endoreplicative process is controlled by factors that are nodule-specific
cysteine-rich (NCR) peptides generated from the host plant and targeted to the bacterial
periplasm, with the ability to penetrate the bacteria membrane and function in its cytoplasm
(Van de Velde et al., 2009).

8. Future perspectives
There are many details pending even in the best studied bacterial models. Some of the
advantages of knowing in detail the replication process and its regulation are the
possibilities for controlling the replication rates in bacteria, for example, to block the DNA
replication of a pathogen or achieve cell-synchronization in bacterial cultures. Using this last
premise, Ferullo et al. (2009), developed a method for synchronizing E. coli cultures, by
treating the bacteria with DL-serine hydroxamate, a structural analogue of the amino acid
serine, this treatment induces a natural stringent response, causing the arrest of the initiation
of DNA replication, once the stringent signal is released, cells initiate a synchronized round
of DNA replication.
Another advantage of knowing the details of the replication process and its regulation is to
allow us to control and use it as a clock in some bio-engineered systems, an example of this
is the ON and OF switch, generated by the methylated or hemimethylated state of DNA in
E. coli (Low & Casadesús, 2008), specially at the GATC sites of the regulatory regions of
many genes and the possibility of timing the replication rate in this organism.
Some organisms with reduced genomes such as the obligate endosymbionts Baumannia
cicadellinicola and Carsonella ruddii, have lost most of the relevant components of the
replicative machinery, such as DnaA. It is suggested that, the lack of DnaA allows the host
to control DNA replication of the symbiont avoiding over-reproduction of the bacteria in its
cytosol (Akman et al., 2002). Another possibility is that DNA replication happens at a low
basal- rate in these stable conditions, in an unrepressed manner. It is postulated that the
association between different organisms leads to adaptation in the rate of DNA replication
of the bacteria in balance with the developmental status of their hosts (Gil et al., 2003).

9. Conclusions
The replication of DNA is a complex process in which a great number of regulators and
mechanisms are involved, one of the most important is the DnaA protein. Replication
normally begins by the formation of a complex of DnaA at the oriC region, with the
assistance of DiaA, and the incorporation of some proteins that form the replisome,
subsequently the formation of the open complex takes place, followed by a complex
238 Fundamental Aspects of DNA Replication

interaction of the proteins needed to execute and complete the DNA replication. The process
finalizes with the recognition of the ter site and disassembly of the replisome. Many of the
proteins are broadly conserved within the bacteria but some special factors are required in
bacteria which undergo particular processes such as asymmetrical cell division.
In general these processes are controlled by a series of circuits, which usually center on the
oriC and affect the activity of DnaA. The result is regulation of the initiation step of DNA
replication. Some of the regulatory mechanisms are time-dependent allowing only one DNA
replication event per cell cycle. The methylation state of the DNA-strands is another
important condition that not only controls the possibility of starting DNA replication but
also regulates the transcription of many genes important for the execution of this function.
All or certain of these mechanisms are adjusted under some special conditions, such as
when the stringent response is triggered by amino acid starvation. In some bacteria with
extremely reduced genomes it is still a mystery as to how DNA replication takes place and
how it is controlled. Many of these latter organisms lack several important proteins
implicated in the control and execution of DNA replication, and these bacteria can be useful
as models for generating a system with the minimal components necessary for DNA
replication.

10. Acknowledgments
Authors thank June Simpson for critical comments to the ms and David Velázquez Ramírez
for assistance with Figures 1-5. This work was supported by CONACYT grant (102854) for
young researchers given to AM-A. CQ-V thanks CINVESTAV-IPN and UAA for a PhD
scholarship and LE-S thanks CONACYT for a PhD scholarship (208153).

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